Histone deacetylase (HDAC) proteins are a family of enzymes that control the acetylation state of protein lysine residues, notably lysine residues contained in the N-terminal extensions of core histones. The acetylation state of histones affect gene expression by influencing chromatin conformation. In addition, the stability or biological function of several non-histone proteins is regulated by the acetylation state of specific lysine residues (Gallinari et al., 2007, Cell Res. 17:191-211; Kazantsev and Thompson, 2008, Nat Rev Drug Discov. 7:854-868).
In humans, HDAC proteins comprise a family of 18 members, which are separated into four classes based on size, cellular localization, number of catalytic active sites, and homology to yeast HDAC proteins. Class I includes HDAC1, HDAC2, HDAC3, and HDAC8. Class II consists of six HDAC proteins that are further divided into two subclasses. Class IIa includes HDAC4, HDAC5, HDAC7, and HDAC9, which each contain a single catalytic active site. Class IIb includes HDAC6 and HDAC10, which each contain two active sites, although only HDAC6 has two catalytically competent active sites. HDAC11 is the sole member of class IV, based on phylogenetic analysis. Class I, II, and IV HDAC proteins operate by a metal ion-dependent mechanism, as indicated by crystallographic analysis. In contrast, class III HDAC proteins, referred to as sirtuins (i.e., SIRT1 through SIRT7), operate by a NAD+-dependent mechanism unrelated to the other HDAC proteins (Gregoretti et al., 2004, J Mol Biol. 338:17-31; Grozinger and Schreiber, 2002, Chem Biol. 9:3-16).
The overexpression of different isoforms of HDACs has been found in several types of cancers, as well as in neurological and inflammatory pathologies. The use of HDAC inhibitors represents a treatment for such diseases (Valente and Mai, 2014, Expert Opin. Ther. Patents, 24:1-15; Falkenberg and Johnstone, 2014, Nat Rev Drug Discov. 13:673-91). The following are examples of HDAC inhibitors that have been tested in clinical trials both as single agents and in combination with chemotherapies and other targeted therapeutics: ACY1215 (Acetylon), CG200745 (Crystal Genomics), 4SC-202 (4SC corporation), CHR-2845 (Chroma Therapeutics), AR-42 (Arno Therapeutics), CUDC-101 (Curis Inc), Givinostat (Italfarmaco), Resminostat (4SC-Corporation), Pracinostat (S*BIO Pte Ltd), Etinostat (Syndax), Abexinostat (Pharmacyclics), Mocetinostat (Methylgene), Belinostat (TopoTarget), Valproic Acid (Instituto Nacional de Cancerologia), Panobinostat (Novartis), Vorinostat (Merck), and Romidepsin (Celgene).
HDAC inhibitors have been combined with a broad range of agents (Bots, & Johnstone, 2009. Clin. Cancer Res. 15, 3970-3977). The most prominent example of the empirical testing of HDAC inhibitors in combination is with DNA-damaging chemotherapeutics, which have led to many successful outcomes (Thum, et al, 2011, Future Oncol. 7, 263-283). HDAC inhibitors have also been successfully combined with DNMT inhibitors. Two Phase I trials have been carried out with vorinostat and bortezomib for the treatment of relapsing and/or refractory multiple myeloma with overall positive responses (Weber D M, Graef T et al 2012, Clin. Lymphoma Myeloma Leuk. 12, 319-324). A Phase III trial is currently assessing VPA (Valproic acid) in combination with levocamitine in children with spinal muscular atrophy (ClinicalTrials.gov identifier: NCT01671384). Vorinostat, panobinostat and VPA are currently being tested in combination with various antiretroviral therapies (ClinicalTrials.gov identifiers: NCT01680094, NCT01319383 and NCT01365065). A Phase I study combining Panobinostat with Ipilimumab to treat unresectable III/IV melanoma has just started (ClinicalTrials.gov identifiers: NCT02032810). HDAC6-specific inhibitors, rocilinostat (ACY-1215), is being tested clinically for the treatment of multiple myeloma in combination with bortezomib, following promising preclinical results (Santo L, Hideshima T, et al, 2012. Blood; 119: 2579-2589.).
Many of the earlier HDAC inhibitors tested in clinical trials are either pan-inhibitors or have poor isoform selectivity. Thus, there is an interest in identifying HDAC inhibitors exhibiting selectivity within or between the human HDAC isoform classes. Achieving selectivity would not only reduce side effects, but would also provide the ability to target distinct therapeutic areas (Hu et al., 2003, J Pharmacol. Ther. 307: 720-728; Giannini et al., 2012, Future Med Chem. 4:1439-1460; Weïwer et al., 2013, Future Med Chem. 5:1491-1508; Falkenberg and Johnstone, 2014, Nat Rev Drug Discov. 13:673-91).
HDAC6 is a well-characterized class IIb deacetylase that regulates many important biological processes via the formation of complexes with its partner proteins. HDAC6 possesses two catalytic domains and a C-terminal zinc finger domain (ZnF-UBP domain, also known as BUZ) that binds free ubiquitin, as well as mono and polyubiquitinated proteins, with high affinity. HDAC6 is localized predominantly in the cytoplasm, and has been reported as a tubulin deacetylase that has effects on microtubule (MT)-mediated processes through both deacetylase-dependent and deacetylase-independent mechanisms. HDAC6 is important both for cytoplasmic and nuclear functions. Unlike other deacetylases, HDAC6 has unique substrate specificity for non-histone proteins such as α-tubulin, HSP90, cortactin, peroxiredoxins, chaperone proteins, β-Catenin, and hypoxia inducible factor-1α (HIF-1α) (Blackwell et al., 2008, Life Science 82:1050-1058; Shnakar and Sirvastava, 2008, Adv Exp Med Biol 615:261-298). HDAC6 also deacetylates protein peroxiredoxins, which are proteins critical in protecting cells from the oxidative effects of H2O2 (Parmigiani et al., 2008, PNAS 105:9633-9638). However, HDAC6 does not catalyze histone deacetylation in vivo. Therefore, it is a safer drug target since it does not impact DNA biology. As a MT-mediated cytoplasmic enzyme, HDAC6, through complexes with partner proteins, regulates multiple important biological processes, such as cell migration, cell spreading, immune synapse formation, viral infection, the degradation of misfolded proteins and stress granule (SG) formation. Mice lacking HDAC6 are viable and have greatly elevated tubulin acetylation in multiple organs. In addition, mice lacking HDAC6 exhibit a moderately impaired immune response and bone homeostasis. Such diverse functions of HDAC6 suggest that HDAC6 serves a potential therapeutic target for the treatment of a wide range of diseases. HDAC6 selective inhibitors have been tested in preclinical indications for cancers, neurology, inflammation, Gaucher's disease, Parkinson's disease, Huntington's disease; Alzheimer's diseases, depression and anxiety, and pain etc. (Gianniniet et al., 2012, Future Med Chem. 4:1439-1460; Falkenberg and Johnstone, 2014, Nat Rev Drug Discov. 13:673-91;).
HDAC8, on the basis of sequence homology, is considered to be a class I enzyme, although phylogenetic analysis has shown it to lay near the boundary of the class I and class II enzymes. HDAC8's importance has been revealed by knockdown experiments of selective HDAC isoforms showing it as essential for cell survival. HDAC8 specific inhibition selectively induces apoptosis in T-cell derived lymphoma and leukemic cells The expression of HDAC8 has been described in a variety of cancer entities e.g. colon, breast lung, pancreas and ovary cancer (Nakagawa et al. 2007, Oncol Rep, 18:769-774). In the highly malignant childhood cancer neuroblastoma high HDAC8 expression significantly correlates with poor prognostic markers and poor overall and event-free survival. In cultured neuroblastoma cells knockdown and pharmacological inhibition of HDAC8 resulted in inhibition of proliferation, reduced clonogenic growth, cell cycle arrest and differentiation (Oehme et al. 2009, Clin Cancer Res, 15:91-99). Furthermore, HDAC8 promotes lung, colon and cervical cancer cell proliferation and may regulate telomerase activity. The three dimensional crystal structure of human HDAC8 was the first to be solved, and 14 human HDAC8 structures co-crystallized with different inhibitors have been described. Currently, HDAC 8 selective inhibitors are in preclinical trials for cancer (Giannini G et al., 2012, Future Med Chem. 4:1439-1460; Falkenberg and Johnstone, 2014, Nat Rev Drug Discov. 13:673-91). Thus, there remains a need in the art for inhibitors of HDACs having high selectivity within and between various HDAC classes, which can serve as therapeutic agents against a variety of diseases and disorders. The present invention fulfills this need.
Class I HDACs, including HDAC1, HDAC2, HDAC3 and HDAC8, are regulating cell survival and proliferation, which makes them ideal target for a variety of cancer types. In addition, HDAC1, HDAC2 and HDAC3 play important roles in regulating learning and memories (Mottamal M et al., 2015, Molecules 20:3898-3941). HDAC2 negatively regulates learning and memory (Guan J S et al., 2009, Nature, 459: 55-60). In mature neurons, the upregulated level of HDAC2 affects the basic excitatory neurotransmission, implying that HDAC2 plays a role in synaptic plasticity (Akhtar M W et al, 2009, J. Neurosci, 29:8288-8297). Knockout and/or over-expression transgenic mouse models of HDAC2, HDAC3 and HDAC6 have demonstrated that loss of function of these individual isoforms can enhance memory and synaptic plasticity (Guan J S et al., 2009, Nature, 459: 55-60; McQuown S C et al., 2010, Curr. Psychiatr. Rep., 12:145-153; Morris M J Et al, 2013, J. Neurosci., 33:6401-6411). HDAC2 inhibitors have been evaluated as therapeutic agents for neurological disorders such as Alzheimer's, Parkinson's, PTSD (Post Traumatic Stress Disorder) etc. (Graff J et al, 2012, Nature, 483:222-226; Graff J. Et al, 2014, Cell 156:261-276).
Sirtuins 1-7 (SIRT1-7) belong to the third class of deacetylase enzymes, which are dependent on NAD(+) for activity. Sirtuins activity is linked to gene repression, metabolic control, apoptosis and cell survival, DNA repair, development, inflammation, neuroprotection, and healthy aging. Because sirtuins modulation could have beneficial effects on human diseases there is a growing interest in the discovery of small molecules modifying their activities. Sirtuin inhibitors with a wide range of core structures have been identified for SIRT1, SIRT2, SIRT3 and SIRT5 (splitomicin, sirtinol, AGK2, cambinol, suramin, tenovin, salermide, among others). SIRT1 inhibition has been proposed in the treatment of cancer, immunodeficiency virus infections, Fragile X mental retardation syndrome and for preventing or treating parasitic diseases, whereas SIRT2 inhibitors might be useful for the treatment of cancer and neurodegenerative diseases. (Villalba et al 2012, 38(5):349-59; Chen L, Curr Med Chem. 2011; 18(13):1936-46).
Thus, there remains a need in the art for inhibitors of HDACs having high selectivity within and between various HDAC classes, which can serve as therapeutic agents against a variety of diseases and disorders. The present invention fulfills this need.